Method and composition for treating acne

ABSTRACT

The subject invention provides a method of treating acne in a subject which comprises topically and periodically applying to the subject&#39;s acne a composition comprising 3-phenyl-benzo[f]chromen-1-one and a pharmaceutically acceptable carrier, wherein the 3-phenyl-1-benzo[f]chromen-1-one is present in an amount effective to treat the subject&#39;s acne. The subject invention also provides a method of treating a skin condition associated with abnormal sebum secretion or abnormal sebaceous gland function in a subject, compositions in such methods and a sorting method for identifying agonists of AhR pathway useful in such methods and compositions.

Throughout this application, various publications are referred to byfirst author and year of publication. Full citations for thesepublications are presented in a List of References section immediatelybefore the claims. Disclosures of the publications in the List ofReferences in their entireties are hereby incorporated by reference intothis application in order to more fully describe the state of the art asof the date of the invention described herein.

BACKGROUND

Field of the Invention

The present invention relates to triaging or sorting and selectingsubstances for their capacity for sebosuppressive activity in topicalskin treatments. The invention also concerns substances identified fromsuch triaging or sorting.

The present invention relates more particularly to a topicalpharmaceutical composition for the treatment and/or prevention ofhyperseborrhea and associated seborrheic skin disorders such as acne andseborrheic dermatitis.

DESCRIPTION OF THE PRIOR ART

The aryl hydrocarbon Receptor (AhR) is a transcription factor, whichinduces the expression of some genes while inhibiting the expression ofother genes. A significant number of ligands, both naturally occurringligands present in foods, as well as xenobiotics, have been reported tointeract with this receptor (Abel et al. (2010)). AhR is typicallyexpressed in epithelial and mesenchymal skin cells, as well as in othercell types (Ikuta et al. 2009). International Patent Publications WO2004/041758 and WO 2007/128725 and U.S. Patent Application PublicationNo. 2009/0028804 A1 describe certain in vitro tests to determine theantagonist or agonist nature of such ligands.

The prototypical xenobiotic agonist ligand of the AhR is the notoriousenvironmental toxin 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), betterknown simply as “dioxin” (Mandal (2005)). Other xenotoxic compoundswhich interact as agonists with the AhR are also capable of causingvarious types of damaging tissue lesions. Multiple toxic effects areknown. The most visible are the cystic lesions formerly called“chloracne” but which have more recently been redefined as MADISH(Saurat et al. (2012)). For this reason, the use of AhR pathway agonistsin a therapeutic and/or preventative context as active agents thatbeneficially modulate skin function is counter-intuitive. Indeed,International Patent Publications WO 2004/041758 and WO 2007/128725propose to use AhR antagonists rather than agonists to treat variousdermatological conditions, including acne.

U.S. Patent Application Publication No. 2010/0324109 A1 suggests thatthe application to the skin of certain AhR receptor pathway agonists mayfavorably modulate some skin functions such as sebaceous gland function,acne, defense against infection, wound healing, and skin atrophies whichinvolve dermatoporosis and estrogen deprivation. However, to achieve atherapeutic window between the beneficial application of certain AhRpathway agonists and the detrimental effects of other AHR pathwayagonists, such as TCDD, certain properties must be selected for.

It is currently unknown how to best identify suitable candidates fromAhR agonists that are likely to be endowed with sebosuppressiveproperties and likely to be therapeutically beneficial to man. Currentlyavailable approaches comprise prolonged dosing with atrophy-inducingtests of the differentiated regions of the sebaceous glands in suitableanimal species. These tests involve complex histological interpretationand, in addition, typically require the prolonged chronic application ofthe ligands under consideration.

It is therefore an objective of this invention to propose a triage orsorting method that is rapid, sensitive and predictive of human efficacywith respect to sebosuppressive properties on human skin. This inventionthus provides a novel method to identify sebum reducing AhR pathwayagonists useful to treat certain skin diseases, and novel pharmaceuticalcompositions useful for treating disorders related to abnormalmetabolism mediated by the AhR receptor.

SUMMARY OF THE INVENTION

The subject invention provides a method of treating acne in a subjectwhich comprises topically and periodically applying to the subject'sacne a composition comprising 3-phenyl-1-benzo[f]chromen-1-one and apharmaceutically acceptable carrier, wherein the3-phenyl-1-benzo[f]chromen-1-one is present in an amount effective totreat the subject's acne.

The subject invention also provides a method of treating a skincondition associated with abnormal sebum secretion or abnormal sebaceousgland function in a subject which comprises topically and periodicallyapplying to an area of subject's skin affected by the skin condition acomposition comprising 3-phenyl-1-benzo[f]chromen-1-one and apharmaceutically acceptable carrier, wherein the3-phenyl-1-benzo[f]chromen-1-one is present in an amount effective totreat the skin condition.

The subject invention also provides a composition comprising3-phenyl-1-benzo[f]chromen-1-one and a pharmaceutically acceptablecarrier, wherein the 3-phenyl-1-benzo[f]chromen-1-one is present at aconcentration between about 0.005% and about 5% by weight.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 provides illustrations (1A, 2A, 3A and 4A) identifying thelocation inside a sebaceous gland of the four types of sebaceous cellsable to express the CYP1A1 gene, namely:

the progenitor cells (1A);

the undifferentiated cells (2A);

the differentiated cells (3A); and

the mature cells (4A).

FIG. 2 shows photomicrographs (1B and C, 2B, 3B and 4B) identifying thefour types of cells able to express the CYP1A1 gene, following treatmentwith Ahr pathway agonists. 1B and C show progenitor cells followingadministration of NSA4; 2B shows undifferentiated cells followingtreatment with NSA1; 3B shows differentiated cells following treatmentwith NSA2 and 4B shows mature cells following treatment with NSA3.Photograph C shows progenitor cells at a different viewing angle andmagnification of the region shown in photograph 1B.

FIG. 3 shows the onset of CYP1A1 activity vs. time in all foursub-populations of sebaceous cells from FIGS. 1 and 2, after topicalapplication of several ligands of the AhR receptor. In this figure, thebiological effect is defined as the stage of sebaceous celldifferentiation, signified by values of 1 to 4 corresponding to stage 1to 4 of differentiation as discussed above; these effects are affectedby exposure to test compounds as indicated in visualization of CYP1A1.

FIG. 4 shows the inhibition of the transcription of certain genesencoding sebogenic enzymes by three structurally related compounds(NSA-2, NSA-8 and NSA-9) and compared with NSA-3(TCDD).

FIG. 5 shows the relative surface occupied by the sebaceous glands inthe dermis over time, subsequent to treatment with NSA-2(3-phenyl-1-benzo[f]chromen-1-one) at three different concentrations.

FIG. 6 shows the differentiation index in the sebaceous glandssubsequent to treatment with NSA-2 at three different concentrations fora 3 week period (5 application days per week).

FIG. 7 shows the number of differentiated and mature sebocytessubsequent to treatment with NSA-2 at three different concentrations.

FIG. 8 shows the number of active sebaceous glands subsequent totreatment with NSA-2 at three different concentrations.

DETAILED DESCRIPTION OF THE INVENTION

The subject invention provides a method of treating acne in a subjectwhich comprises topically and periodically applying to the subject'sacne a composition comprising 3-phenyl-1-benzo[f]chromen-1-one and apharmaceutically acceptable carrier, wherein the3-phenyl-1-benzo[f]chromen-1-one is present in the composition in anamount effective to treat the subject's acne.

In one embodiment, 3-phenyl-1-benzo[f]chromen-1-one, also known asbeta-naphthoflavone, is present in the composition at a concentrationbetween about 0.005% and about 5% by weight. In another embodiment,3-phenyl-1-benzo[f]chromen-1-one is present in the composition at aconcentration between about 0.1% and about 2.5% by weight. In yetanother embodiment, 3-phenyl-1-benzo[f]chromen-1-one is present in thecomposition at a concentration of about 0.1% by weight. In still otherembodiments, 3-phenyl-1-benzo[f]chromen-1-one is present in thecomposition at a concentration of about 0.25% by weight, about 0.5% byweight, about 1% by weight, about 2% by weight, or about 5% by weight.

In some embodiments, the pharmaceutically acceptable carrier comprisesethanol.

In other embodiments, the pharmaceutically acceptable carrier comprisespolyethylene glycol having an average molecular weight between 200 g/moland 1000 g/mol. In one such embodiment, the polyethylene glycol has anaverage molecular weight of about 400 g/mol.

In yet other embodiments, the pharmaceutically acceptable carriercomprises a mixture of ethanol and polyethylene glycol in a ratio from5:1 to 1:5 by volume, for example, between 2:1 and 1:2 by volume,particularly about 1:1 by volume.

In some embodiments, the pharmaceutical composition is a solution andcomprises 3-phenyl-1-benzo[f]chromen-1-one at a concentration between0.005 g and 1.0 g 3-phenyl-1-benzo[f]chromen-1-one per 100 mL of thecomposition and the pharmaceutically acceptable carrier comprises amixture of ethanol and polyethylene glycol having an average molecularweight of about 400 g/mol in a ratio of about 1:1 by volume. In anotherembodiment, the concentration of 3-phenyl-1-benzo[f]chromen-1-one isbetween 0.05 g and 0.5 g. In yet another embodiment, the3-phenyl-1-benzo[f]chromen-1-one is at a concentration of about 0.5 g,the polyethylene glycol has an average molecular weight of about 400g/mol and the mixture of ethanol and polyethylene glycol is in a ratioof about 1:1 by volume.

In certain embodiments, the pharmaceutically acceptable carrier furthercomprises one or more of an alcohol, an anti-bacterial agent, apreservative, and a chelating agent.

In some embodiments, the pharmaceutical composition is in the form of alotion, gel, cream, ointment, foam, solution, suspension, dispersion orimpregnated dressing.

In some embodiments, the acne is facial acne; in other embodimentschest, back and/or shoulder acne, for example, the acne associated withPropionibacterium acnes or the acne is associated with a high sebumsecretion rate.

In some embodiments, 3-phenyl-1-benzo[f]chromen-1-one is topicallyapplied daily. In other embodiments, 3-phenyl-1-benzo[f]chromen-1-one istopically applied only at night. In still other embodiments,3-phenyl-1-benzo[f]chromen-1-one is topically applied twice or threetimes daily. In still further embodiments,3-phenyl-1-benzo[f]chromen-1-one is topically applied every other day.In still other embodiments, 3-phenyl-1-benzo[f]chromen-1-one istopically applied weekly.

The subject invention also provides a method of treating a skincondition associated with abnormal sebum secretion or abnormal sebaceousgland function in a subject which comprises topically and periodicallyapplying to an area of the subject's skin affected by the skin conditiona composition comprising 3-phenyl-1-benzo[f]chromen-1-one and apharmaceutically acceptable carrier, wherein the3-phenyl-1-benzo[f]chromen-1-one is present in the composition in anamount effective to treat the skin condition.

The skin condition may be any of oily skin, oily hair, shiny orgreasy-looking skin, hyperseborrhea, seborrheic dermatitis, rosacea,sebaceous hyperplasia or sebaceous carcinoma. In some embodiments, theskin condition is seborrheic dermatitis. In other embodiments, the skincondition is rosacea. In yet other embodiments, the skin condition ishyperseborrhea, sebaceous hyperplasia, or sebaceous carcinoma.

In some embodiments, 3-phenyl-1-benzo[f]chromen-1-one is present in thecomposition at a concentration between about 0.005% and about 5% byweight. In other embodiments, 3-phenyl-1-benzo[f]chromen-1-one ispresent in the composition at a concentration between about 0.1% andabout 2.5% by weight. In yet other embodiments,3-phenyl-1-benzo[f]chromen-1-one is present in the composition at aconcentration of about 0.1% by weight, about 0.25% by weight, about 0.5%by weight, about 1% by weight, about 2% by weight, or about 5% byweight.

In some embodiments, the pharmaceutically acceptable carrier comprisesethanol.

In other embodiments, the pharmaceutically acceptable carrier comprisespolyethylene glycol having an average molecular weight between 200 g/moland 1000 g/mol. In one such embodiment, the polyethylene glycol has anaverage molecular weight of about 400 g/mol.

In yet other embodiments, the pharmaceutically acceptable carriercomprises a mixture of ethanol and polyethylene glycol in a ratio from5:1 to 1:5 by volume, for example, between 2:1 and 1:2 by volume,particularly about 1:1 by volume.

In some embodiments, the pharmaceutical composition is a solution andcomprises 3-phenyl-1-benzo[f]chromen-1-one at a concentration between0.005 g and 1.0 g 3-phenyl-1-benzo[f]chromen-1-one per 100 mL of thecomposition and the pharmaceutically acceptable carrier comprises amixture of ethanol and polyethylene glycol having an average molecularweight of about 400 g/mol in a ratio of about 1:1 by volume. In anotherembodiment, the concentration of 3-phenyl-1-benzo[f]chromen-1-one isbetween 0.05 g and 0.5 g. In another embodiment, the ratio is between2:1 and 1:2 by volume. In one such embodiment, the3-phenyl-1-benzo[f]chromen-1-one is at a concentration of about 0.5 g,the polyethylene glycol has an average molecular weight of about 400g/mol and the mixture of ethanol and polyethylene glycol is in a ratioof about 1:1 by volume.

In certain embodiments, the pharmaceutically acceptable carrier furthercomprises one or more of an alcohol, an anti-bacterial agent, apreservative, and a chelating agent.

In some embodiments, the pharmaceutical composition is in the form of alotion, gel, cream, ointment, foam, solution, suspension, dispersion orimpregnated dressing.

In some embodiments, the area of the subject's skin affected by the skincondition is on the face, chest, shoulders or back. In otherembodiments, the skin condition is associated with Propionibacteriumacnes and/or a high sebum secretion rate.

In some embodiments, 3-phenyl-1-benzo[f]chromen-1-one is topicallyapplied daily. In other embodiments, 3-phenyl-1-benzo[f]chromen-1-one istopically applied only at night. In still other embodiments,3-phenyl-1-benzo[f]chromen-1-one is topically applied twice or threetimes daily. In still further embodiments,3-phenyl-1-benzo[f]chromen-1-one is topically applied every other day.In still other embodiments, 3-phenyl-1-benzo[f]chromen-1-one istopically applied weekly.

The subject invention also provides a composition comprising3-phenyl-1-benzo[f]chromen-1-one and a pharmaceutically acceptablecarrier, wherein the 3-phenyl-1-benzo[f]chromen-1-one is present at aconcentration between about 0.005% and about 5% by weight.

In some embodiments, the 3-phenyl-1-benzo[f]chromen-1-one is present ata concentration between about 0.1% and about 2.5% by weight. In otherembodiments, the 3-phenyl-1-benzo[f]chromen-1-one is present at aconcentration of about 0.1% by weight. In yet other embodiments, the3-phenyl-1-benzo[f]chromen-1-one is present at a concentration of about0.25% by weight, about 0.5% by weight, about 1% by weight, about 2% byweight, or about 5% by weight.

In some embodiments, the pharmaceutically acceptable carrier comprisesethanol.

In other embodiments, the pharmaceutically acceptable carrier comprisespolyethylene glycol having an average molecular weight between 200 g/moland 1000 g/mol. In one such embodiment, the polyethylene glycol has anaverage molecular weight of about 400 g/mol.

In other embodiments, the pharmaceutically acceptable carrier comprisesa mixture of ethanol and polyethylene glycol in a ratio from 5:1 and 1:5by volume, for example, between 2:1 and 1:2 by volume, particularly,about 1:1 by volume.

In some embodiments, the pharmaceutical composition is a solution andcomprises 3-phenyl-1-benzo[f]chromen-1-one at a concentration between0.005 g and 1.0 g 3-phenyl-1-benzo[f]chromen-1-one per 100 mL of thecomposition and the pharmaceutically acceptable carrier comprises amixture of ethanol and polyethylene glycol (having an average molecularweight of about 400 g/mol in a ratio of about 1:1 by volume. In otherembodiments, the concentration of 3-phenyl-1-benzo[f]chromen-1-one isbetween 0.05 g and 0.5 g. In one such embodiment, the3-phenyl-1-benzo[f]chromen-1-one is at a concentration of about 0.5 g,the polyethylene glycol has an average molecular weight of about 400g/mol and the mixture of ethanol and polyethylene glycol is in a ratioof about 1:1 by volume.

In certain embodiments, the pharmaceutically acceptable carrier furthercomprises one or more of an alcohol, an anti-bacterial agent, apreservative, and a chelating agent.

In some embodiments, the pharmaceutical composition is in the form of alotion, gel, cream, ointment, foam, solution, suspension, dispersion orimpregnated dressing.

The subject invention also provides a method of predicting clinicalresponsiveness of a subject to treatment of acne by topical applicationof 3-phenyl-1H-benzo[f]chromen-1-one, the method comprising inducingCYP1A1 expression in the subject and evaluating the amount of CYP1A1expressed so as to predict the clinical responsiveness of the subject.

In certain embodiments, the amount of CYP1A1 expressed has a positivecorrelation with clinical responsiveness.

The subject invention also provides a method of predicting clinicalresponsiveness of a subject to treatment of a skin condition associatedwith abnormal sebum secretion or abnormal sebaceous gland function bytopical application of 3-phenyl-1H-benzo[f]chromen-1-one, the methodcomprising inducing CYP1A1 expression in the subject and evaluating theamount of CYP1A1 expressed to predict the clinical responsiveness of thesubject.

In some embodiments, the skin condition is oily skin, oily hair, shinyor greasy-looking skin, hyperseborrhea, seborrheic dermatitis, rosacea,sebaceous hyperplasia or sebaceous carcinoma.

In certain embodiments, the amount of CYP1A1 expressed has a positivecorrelation with clinical responsiveness.

The subject also relates to a method of triaging or sorting andselecting substances in order to better determine their capacity forsebosuppressive activity in topical or local skin treatments, comprisingan in vivo test, the said in vivo test comprising the following steps:

-   -   a) choosing a substance from amongst sebum reducing AhR pathway        agonists;    -   b) choosing a mammal in which the CYP1A1 gene can be induced in        the skin;    -   c) treating a part of the skin of said mammal, chosen in        relation to the localization of sebaceous glands thereof, via a        topical route, with a composition containing the said substance,        following a dose response vs. time protocol;    -   d) examining by means of immune-histological staining the        expression of CYP1A1 in the sebaceous glands of the chosen said        mammal treated locally on the skin; and    -   e) selecting said substance in relation to the sequence of onset        of immunohistochemical labelling vs. time in several different        types (compartments) of cells of the said sebaceous glands

The said sebum reducing AhR pathway agonists to be tested are preferablychosen from among known AhR agonists or first determined to be an AhRagonist by at least one suitable in vitro test, for example firstscreening using the CALUX (He et al. 2011) and/or EROD tests(Zamaratskaia et al. (2009), Behnisch et al. (2001), to determine bothpotency and the degree of maximum induction, combined with theunderstanding or demonstration of a short in vivo half-life by standardmethods known to those skilled in the art.

For testing in vivo, before man, rodents, in particular, the mouse, arepreferred mammals for this purpose, more preferably the murine C57BL/6strain.

As far as the site of application of the compounds to rodents isconcerned, the skin of the ears, which are known to contain multiplesebaceous glands, are particularly preferred. Being well suited to thistype of analysis they are also a locale where the CYP1A1 gene is likelyto be induced.

According to a further embodiment, the ears of the said mice are treatedvia the topical route, then sampled vs. time, and the expression ofCYP1A1 is examined by immunohistochemical analysis using an antibody. Inparticular, but not limited thereto, the antibody used may be the rabbitanti-rat CYP1A1 polyclonal antibody (Millipore AB1247).

In such an embodiment, the examination of the expression of CYP1A1 inthe sebaceous glands may comprise:

-   -   a) examination of the isthmus region, in particular examination        of the progenitor cells (see Jensen et al. 2009, Niemann et al.        2012);    -   b) examination of the peripheral regions of the gland, in        particular examination of the undifferentiated cells;    -   c) examination of the intermedia region of the gland, in        particular examination of the differentiated cells; and/or    -   d) examination of the central region of the gland, in particular        examination of the mature cells.

The substance is considered active if the said in vivo test exhibitsimmunohistochemical staining in the plurality of relevant cell typesindicated above within a determined time period; After examining thesefour cell types, the said substance can be selected if the expression ofCYP1A1 is labeled in at least two cell types after one week's treatment.

After examining these four cell types, the said substance is preferablyselected if the expression of CYP1A1 is labelled in all four cell typesafter one week's treatment.

Further selection can be made by treating said mammal with a potentialsebum reducing AhR pathway agonist and measuring markers of actual sebumproduction by standard methods, including squalene and/or waxy esters(e.g., Smith et al. (2008), Miyazaki et al. (2001)).

According to a further embodiment of the present invention, an object ofthe invention is a composition for treating and/or preventing skindiseases of a human being, in particular the associated skin conditionsof acne, seborrheic dermatitis and rosacea, wherein the composition isable to treat and/or prevent hyperseborrhea by means of topical or localapplication of said composition on the skin, and composition comprisingan active substance selected from the group consisting of AhR pathwayagonists having:

-   -   a) an ability to activate one or more components of the sebum        reducing AhR pathway;    -   b) an ability to modulate a gene regulated by the AhR pathway;    -   c) a short half-life in the human organism, either predicted        from standard mammalian pharmacokinetic studies or actually        determined, of less than 24 hours; preferably less than 4 hrs    -   d) a measurable positive effect on a recognized criterion of        sebum reduction.

A presently preferred embodiment of the invention is rutecarpine, apharmaceutically acceptable salt of rutecarpine, or an herbal(plant/fruit) extract comprising rutecarpine as the active substance ina sebosuppressive composition for topical use.

Another presently preferred embodiment of the invention is3-phenyl-1H-benzo[f]chromen-1-one, as the active substance in asebosuppressive composition for topical use.

According to yet a further aspect of the invention, an object of theinvention is a method for treating and/or preventing skin diseases of ahuman being, such as acne, seborrheic dermatitis and rosacea, comprisingproviding a composition that is able to treat and/or preventhyperseborrhea by means of topical application of said composition onthe skin, said composition comprising an active substance selected fromthe group consisting of sebum reducing AhR pathway agonists having:

-   -   a) an ability to act as a sebum reducing AhR pathway agonist;    -   b) an ability to modulate a gene regulated by the AhR pathway;    -   c) a short half-life in the human organism, either predicted        from standard mammalian pharmacokinetic studies or actually        determined of less than 24 hours; more preferably less than 4        hours;    -   d) a measurable positive effect on a recognized criterion of        sebum reduction;    -   e) and wherein said active substance is positively selected by        an in vivo test as defined above;    -   f) and wherein said composition is administered topically to        said human being.

A particular object of the invention is thus a process for treatingand/or preventing hyperseborrhea-induced skin conditions in a humanbeing, comprising providing a composition suitable for topicalapplication on the skin, wherein said composition contains3-phenyl-1H-benzo[f]chromen-1-one as the active substance, andadministering said composition topically or locally to said human being.

For all of the foregoing embodiments, each embodiment disclosed hereinis contemplated as being applicable to each of the other disclosedembodiments.

Terms

As used herein, and unless stated otherwise, each of the following termsshall have the definition set forth below.

As used herein, the term “sebaceous glands” refers to microscopic glandsin the skin that secrete an oily/waxy matter, called sebum, to lubricateand waterproof the skin and hair of mammals. In humans, they are foundin greatest abundance on the face and scalp, though they are distributedthroughout all skin sites except the palms and soles.

As used herein, the term “skin” refers to the outer coverage of thebody. In humans, it is the largest organ of the integumentary system.The skin has multiple layers of ectodermal tissue and guards theunderlying muscles, bones, ligaments and internal organs. Human skin issimilar to that of most other mammals, except that it is not protectedby fur. Though nearly all human skin is covered with hair follicles, itcan appear hairless. The adjective cutaneous means “of the skin” (fromLatin cutis, skin).

As used herein, the term “dermatoporosis” refers to a new conceptproposed to cover different manifestations and implications of chroniccutaneous insufficiency/fragility syndrome. This emerging syndromeextends beyond cosmetics and appearance and is considered to be thefunctional face of skin aging (Kaya et al. (2007)).

As used herein, the term “acne” refers to acne vulgaris, a common humanskin disease, characterized by areas of skin with seborrhea (scaly redskin), comedones (blackheads and whiteheads), papules (pinheads),nodules (large papules), pimples, papulopustules and possible scarring.Acne affects mostly skin with the densest population of sebaceousglands; these areas include the face, the upper part of the chest, andthe back. Severe acne is inflammatory, but acne can also manifest innon-inflammatory forms. The lesions are caused by changes inpilosebaceous units, skin structures consisting of hair follicle and itsassociated sebaceous gland, changes that require androgen stimulation.

As used herein, the term EROD refers to the ethoxyreorufin-O-deethylase(EROD) assay which monitors the induction of the xenobiotic-metabolizingenzyme cytochrome P-450 1A1 (CYP1A1) and is a widely used as a reporterfor measuring activation of the AhR in vitro (Zamaratskaia et al.(2009), Behnisch et al. (2001),) As used herein, the term CALUX refersto Chemical-Activated Luciferase Gene Expression (CALUX). The CALUXAssay is a dioxin screening bioassay categorized as a reporter-geneassay. It has been approved as an official analysis method by the US EPAin 2007 (Method 4435) (He et al. (2011))

As used herein, the term CYP1A1 refers to Cytochrome P450, family 1,subfamily A, polypeptide 1. CYP1A1 is a protein that in humans isencoded by the CYP1A1 gene. The protein is a member of the cytochromeP450 superfamily of enzymes, CYP1A1 is involved in phase I xenobioticand drug metabolism (e.g., Monostory et al. (2009), Nerbert and Dalton(2006), Zhou et al. (2009).

As used herein, the term “treating” or “treatment” of any condition,disease or disorder refers, in some embodiments, to ameliorating thedisease, disorder, or condition (i.e., arresting or reducing thedevelopment of the disease, disorder, or condition, or at least one ofthe clinical symptoms thereof). In other embodiments “treating” or“treatment” refers to ameliorating at least one physical parameter,which may or may not be discernible by the subject, including physicalparameters that are undesired but not clinically significant. In yetother embodiments, “treating” or “treatment” refers to inhibiting thedisease, disorder, or condition, either physically, (e.g., stabilizationof a discernible symptom), physiologically, (e.g., stabilization ofphysical parameter) or both. In yet other embodiments, “treating” or“treatment” refers to preventing or to delaying the onset of thedisease, disorder, or condition.

As used herein, the term “therapeutically effective amount” or“effective amount” means the amount of a composition, compound, therapy,or course of treatment that, when administered to a subject for treatinga disease, disorder, or condition, is sufficient to effect suchtreatment for the disease, disorder, or condition. The “therapeuticallyeffective amount” will vary depending on the composition, the compound,the therapy, the course of treatment, the disease, disorder, orcondition, and its severity and the age, weight, etc., of the subject tobe treated.

As used herein, sebum reducing AhR pathway agonists are compounds thatby activating one or more components of the AhR pathway, are capable ofreducing sebum levels in the skin, when applied topically, locally orsystemically.

In the scope of the embodiments, the AhR ligands described hereininclude further forms of the compounds such as pharmaceuticallyacceptable salts, solvates (including hydrates), amorphous phases,partially crystalline and crystalline forms (including all polymorphs),prodrugs, metabolites, N-oxides, isotopically-labeled, epimers, pureepimers, epimer mixtures, enantiomers including but not limited tosingle enantiomers and enantiomeric diastereomers, meso compounds,stereoisomers, racemic mixtures and diasteroisomeric mixtures. AhRligand compounds described herein having one or more double bondsinclude cis/trans isomers, E/Z isomers and geometric isomers. AhR ligandcompounds described herein can be prepared as pharmaceuticallyacceptable salts formed when an acidic proton present in the parentcompound either is replaced by a metal ion, for example an alkali metalion, an alkaline earth ion, or an aluminum ion; or coordinates with anorganic base. AhR ligand compounds described herein can also be preparedas pharmaceutically acceptable salts when a basic function present inthe parent compound coordinates with a mineral acid or an organic acid.AhR ligand compounds can also be described herein as being prepared aspharmaceutically acceptable complexes or co-crystals whereby the complexor co-crystal confers modified physical properties of solubility,dissolution rate or permeability. In addition, the salt forms of thedisclosed compounds can be prepared using salts of the starting materialor intermediates.

In some embodiments, the AhR pathway agonist compounds described hereinare isotopically-labeled, which are identical to those recited in thevarious formulae and structures presented herein, but for the fact thatone or more atoms are replaced by an atom having an atomic mass or massnumber different from the atomic mass or mass number usually found innature. In some embodiments, one or more hydrogen atoms are replacedwith deuterium. In some embodiments, metabolic sites on the compoundsdescribed herein are deuterated.

In some embodiments, substitution with deuterium affords certaintherapeutic advantages resulting from greater metabolic stability, suchas, for example, increased in vivo half-life or reduced dosagerequirements. Throughout the specification, groups and substituentsthereof can be chosen by one skilled in the field to provide stablemoieties and compounds.

This invention will be better understood by reference to theExperimental Details which follow, but those skilled in the art willreadily appreciate that the specific experiments detailed are onlyillustrative of the invention as described more fully in the claimswhich follow thereafter.

EXPERIMENTAL DETAILS Description of an Embodiment of the Triage orSorting Method

According to the state of the art, the biological activity triggered bythe topical or systemic administration of the ligand should be uniformin all parts of the body which express the receptor, provided that theligand is diffused in these parts in sufficient quantity and in anon-metabolized active form, such as seen with the retinoic acidreceptor (RAR) or the vitamin D receptor (Milde et al (1991); Reichrathet al (1997)).

In this case, the application of AhR pathway agonist to the skin shouldactivate the biochemical pathways linked to this receptor uniformly,first in the surface layers of the epidermis, and then progressively,correlating with the entry gradient of the ligand into the deeper layersof the epidermis, possibly the dermis and adnexa, hairs and sebaceousglands. It is known that the AhR receptor is expressed in all thesecompartments of the skin (Ikuta et al. 2009).

Yet, surprisingly, the applicant has found that the topical applicationto the skin of a sebum reducing AhR pathway agonist which is capable ofacting as a sebosuppressive agent, activates the pathway of thisreceptor in focal manner on the sebaceous glands starting with theprogenitor cells, i.e. the sebaceous stem cells located in the isthmicregion of the pilosebaceous unit, followed in sequence by thenon-differentiated sebaceous cells, the differentiated sebaceous cells,and finally the mature cells. These observations are illustrated inFIGS. 1 and 2.

Yet more surprisingly, the ligands endowed with the strongestsebosuppressive activity are those which, without necessarily being themost active during in vitro activation tests of the AhR receptors,follow this sequence of activation in situ at an early stage, rapidlyand completely as shown by Tables 1 and 2.

These observations are counter-intuitive to those of M. J. Rowe et al.(Rowe et al. (2008)) who have shown that in a transgenic mouse with aCYP1A1-GFP promoter the biological activity induced by an AhR pathwayagonist administered via the systemic route could be localized to thesebaceous glands, but in a uniform manner within the latter:

-   -   a) First, according to the applicant's observations, the        administration route is transcutaneous, and the order of        distribution is fully unexpected according to the sequence of        transepidermal diffusion explained above. In the work conducted        by Rowe et al., the ligand, 3-methylcholanthrene, is        administered via the systemic route which results in access to        the skin via the blood. The distribution of a lipophilic ligand        in the sebaceous gland in therefore not surprising;    -   b) Second, and more specifically, the essential activation        sequence of the present invention, i.e. progenitor        cells>undifferentiated cells>differentiated cells>mature cells,        was not observed by Rowe et al. (Rowe et al. (2008)) who        demonstrate diffuse biological activity in the region of the        sebaceous glands. The work of Rowe, et al. would not have        allowed either the determination or the suggestion of the method        for triaging/sorting sebosuppressive ligands which is the        subject of this invention.    -   c) Third, and more specifically, Rowe et al. did not measure        sebum levels or make any measurement of lipid production.    -   d) Fourth, Rowe et al. did not mention either        3-phenyl-1-benzo[f]chromen-1-one or rutecarpine or suggest their        use in the treatment of acne.

Demonstration of the Sequential Activation of Sebum Reducing AhR PathwayAgonists in the Focal Regions of the Sebaceous Glands

The embodiment described here entails treating the ears of C57BL/6 micevia the topical route, following established protocols to determinedose-response and time-response relationships, with sebum reducing AhRpathway agonists previously characterized for their in vivo activationof the receptor, using, for example the EROD and CALUX methods which arewidely used in this field (see Table 1).

TABLE 1 Sebo- CALUX EROD Conc. genesis Efficiency Efficiency testedFocal expression of AhR inhibition % of % of in the activation (CYP1A1)index in 10 nM 10 nM Mouse Progenitor Undiffer- Differ- mice Com- Conc.TCDD Conc. TCDD Model cells entiated entiated Mature % Human pound μMefficiency μM efficiency mM* Stage 1 Stage 2 Stage 3 Stage 4 controlactivity NSA 1 10 78 1 25 12

 

 

 

 

 

 

 

 

28

NSA 2 5 13 10 20 45

 

 

 

 

 

 

 

 

90

 

 

NSA 3 0.01 100 0.01 100 0.0062

 

 

 

 

 

 

 

 

97

 

 

NSA 4 0.1 76 0.1 60 0.035

 

 

 

 

 

 

 

 

8 NT NSA 5 100 32 100 30 41

 

 

 

 

 

 

 

 

11 NT NSA 6 100 58 100 10 21

 

 

 

 

 

 

 

 

16 NT NSA 7 100 17 232

 

 

 

 

 

 

 

 

5

NT = NOT TESTED

 

 

 and

 = No activity

 

 

 Active

 Weak activity *corresponding to 1% in acetone NSA-1 and NSA-2 were bothtested in man at 0.5% NSA-7 was tested in man at 8%.

Table 1 shows the correlations between in vitro tests, in vivo testsaccording to the invention, and clinical examination of thesubosupressive activity in man of the ligands from FIG. 3.

Following topical treatment, the ears are sampled, and CYP1A1 expressionis examined by immunohistochemical analysis using a specific antibody.

The study diagrammed in FIG. 1 and for which photomicrographs areprovided in FIG. 2 entailed treating mice daily for one week with atopical AhR pathway agonist, with ears taken for examination at Day 7.Ears were recovered and fixed in formalin, paraffin embedded and thensections were prepared and positioned on slides for immunohistochemicalstaining. The specific antibody used in this instance was rabbitanti-rat CYP1A1 polyclonal antibody (Millipore AB1247). The AhR pathwayagonists used in the example in FIG. 2 were as follows:

-   -   1B) 6-formylindolo[3,2-b]carbozole (NSA4/FICZ);    -   2B)        8,13-Dihydroindolo[2′,3′:3,4]pyrido[2,1-b]quinazolin-5(7H)-one        (NSA1/rutecarpine);    -   3B) 3-phenyl-1H-benzo[f]chromen-1-one (NSA2);    -   4B) 2,3,7,8-tetrachlorodibenzo-p-dioxin (NSA3/TCDD).

NSA2 was used at a concentration of 37 mM, NSA4 was used at 35 μM, NSA1at 12 mM, and NSA3 at 6.2 μM.

Photomicrographs were taken on a Zeiss microscope at a magnification of250-fold for photos 1B, 2B, 3B, 4B of FIG. 2 and at a magnification of400-fold for Part 1C of the Figure.

FIG. 1 shows diagrammatically the different types of labelling observed.A positive result from immunohistochemistry, i.e. the cells stainedbrown as would be shown in color originals of the black and whiteillustrations of FIG. 2, indicates that the region expresses the CYP1A1protein. Results are summarized as follows:

-   -   a) In the basal state the CYP1A1 protein is not detectable.    -   b) In every case in which the AhR pathway agonists were used, it        led to positive staining by CYP1A1 immunohistochemistry. The        first region in time to be stained, indicating an increase in        the CYP1A1 protein induced by activation of the AhR receptor, is        the region of the isthmus where the progenitor cells of the        sebaceous glands are located (FIG. 1-1A and FIG. 2-1B).    -   c) Stage 1 corresponds to multi-potent clonogenic cells, in        other words to the sebaceous stem cells (Frances D and Niemann C        (2012)). In addition to the particular topography of the isthmus        region, these cells are characterized by the expression of        L-RIG1, a marker of isthmic multi-potent clonogenic cells        (Jensen et al. (2009),). Using double CYP1A1 and L-RIG1 staining        it was also possible to show that the cells activated by the        topically administered AhR pathway agonist effectively        correspond to this population at this isthmic region.    -   d) Stage 2 is the extension of CYP1A1 staining to        undifferentiated cells which do not contain lipids and in        general are located on the periphery of the sebaceous gland        (FIG. 1-2A and FIG. 2-2B).    -   e) Stage 3 is the further extension of CYP1A1 staining to        differentiated cells which contain lipids and are in general        located at the intermediate part of the sebaceous gland (FIG.        1-3A and FIG. 2-3B).    -   f) Stage 4 is the further extension of CYP1A1 staining to mature        cells which contain lipids and in general are located at the        central part of the sebaceous gland (FIG. 1-4A and FIG. 2-4B).

Correlation Between the Sequential Activation Stages and theSebosuppressive Properties of the Ligand.

Table 1 indicates the correlation between the stages of focal activationexpression, the index of sebum inhibition, and the effect on human skin.The sebum inhibition index is calculated by counting the number ofmature and differentiated cells in relation to the total number of cellsin the sebaceous glands. A decrease in mature and differentiated cellsindicates blocking of sebogenesis. The effect on human skin isdetermined by sebumetric examination using what is known as the “casuallevel” (Dobrev (2007)).

In FIG. 3 and Tables 1 and 2, the abbreviations used refer to thefollowing chemicals:

-   -   a) NSA1:        8,13-Dihydroindolo[2′,3′:3,4]pyrido[2,1-b]quinazolin-5(7H)-one        (rutecarpine)    -   b) NSA2: 3-phenyl-1H-benzo[f]chromen-1-one    -   c) NSA3: 2,3,7,8-tetrachlorodibenzo-p-dioxin(TCDD)    -   d) NSA4: 6-formylindolo[3,2-b]carbazole(FICZ)    -   e) NSA5:        2-[(E)-2-(3,4-dihydroxyphenyl)ethenyl]-6-hydroxypyran-4-one        (Hispidine)    -   f) NSA6: 9H-β-carboline(Beta-carboline)    -   g) NSA7:        (S)-5-methoxy-2-[(4-methoxy-3,5-dimethylpyridin-2-yl)methylsulfinyl]-3H-benzoimidazole        (Esomeprazole)    -   h) NSA8: 2-phenyl-4H-benzo[h]chromen-4-one    -   i) NSA9: 2-phenyl-4H-chromen-4-one

A comparison between FIG. 3 and Table 1 shows that two AhR pathwayagonists, namely NSA-2 (3-phenyl-1H-benzo[f]chromen-1-one) and NSA-3(TCDD), which activate the AhR pathway in the four cell sub-populationswithin a period of less than one week, are also those which display thestrongest sebosuppressive activity in man. For testing in the mousemodel, all compounds were tested at 1% in acetone. NSA-1 (Rutecarpine),while more effective than NAS-2 in in vitro activation assays, activatesthe AhR pathway in two of the cell sub-populations in mice, also haspotentially useful sebosuppressive activity in man when tested at thesame concentrations (0.5%), although weaker than that of NSA-2. On theother hand, NSA-7, which only activates the AhRP receptor pathways inStage One of the cell sub-populations, shows no sebosuppressive activityin man, even at 8%, even though its ability to activate AhR in vitroappears similar to NSA-2.

Description of Sebosuppressive Property of3-phenyl-1H-benzo[f]chromen-1-one and Related Structural Analogs.

The sebosuppressive activity of 3-phenyl-1H-benzo[f]chromen-1-one wascompared with that of 2-phenyl-4H-benzo[h]chromen-4-one and2-phenyl-4H-chromen-4-one under the same test conditions as thosedescribed above, namely in vitro test, the mouse in vivo tests asdescribed in this invention and by clinical examination in man. Theresults for in vitro activation and focal expression of CYP1A1 in miceare given in Table 2, below. It can be seen that NSA-2(3-phenyl-1H-benzo[f]chromen-1-one) demonstrates agonist activity in thesebaceous glands in a similar manner to the most active AhRP agonist(TCDD), while 2-phenyl-4H-benzo[h]chromen-4-one and2-phenyl-4H-chromen-4-one do not have these effects. Without wishing tobe bound by any theory, it would appear that this capacity for tissuedistribution is somehow dissociated from the agonist activity of themolecule for the AhR receptor as determined by the EROD and CALUXassays, since molecules having much greater activity in those assays,such as NSA4 (FICZ) have limited distribution in the sebaceous glandswhen compared with NSA-2. When the three compounds (NSA-2, NSA-8 andNSA-9) were tested in man at 0.5% neither2-phenyl-4H-benzo[h]chromen-4-one nor 2-phenyl-4H-chromen-4-oneexhibited any reduction in sebum (as determined by sebutape and clinicalobservations). Only NSA-2 was active in reducing sebum. In combinationwith other properties set forth below, this property of sequentiallytargeting the sebaceous glands correlates with the sought-aftertherapeutic effects in humans.

Table 2 shows the activity of TCDD and three other compounds (NSA-2,NSA-8 and NSA-9) in both cell based in vitro tests and in vive testsaccording to the invention.

TABLE 2 CALUX efficiency EROD efficiency Focal expression of AhR % of %of activation in vivo (CYP1A1) Concen- 10 Concen- 10 Non- tration nMtration nM Progenitor differentiated Differentiated Mature (μM) TCDD(μM) TCDD Stage 1 Stage 2 Stage 3 Stage 4 TCDD (NSA-3) 0.01 100 0.01 100

 

 

 

 

 

 

 

 

3-phenyl-1H- 10 (7) 10 20

 

 

 

 

 

 

 

 

benzo[f]chromen- 1-one (NSA-2) 2-phenyl-4H- — — 100 0.7

 

 

 

 

 

 

 

 

benzo[h]chromen- 4-one (NSA-8) 2-phenyl-4H- 100 10 100 11

 

 

 

 

 

 

 

 

chromen-4-one (NSA-9) In mice the three compounds (NSA2, NSA8 and NSA9)were tested at 0.5% in acetone. TCDD (NSA-3) was tested on mice at0.0005%.

 

 

 = No activity

 

 

 = Active

FIG. 4 shows the results of experiments in which the ears of C57BL/6mice were treated with the three different compounds (NSA-2, NSA-8 andNSA-9) above at 0.5% in acetone for five weeks. The reference substance(positive control) was TCDD at a concentration 1000 times lower(0.0005%). At the end of that time the animals were sacrificed and theears removed for further analysis.

To generate the data for FIG. 4, after extraction of RNA by standardmethods, Real-Time QRT-PCR reactions were performed followed by analysisfor the presence of mRNA corresponding to three major enzymes (fattyacid desaturase 2 [FADS2], acyl-CoA wax alcohol acyltransferase 1[AWAT1], and elongation of very long chain fatty acids protein 3 [ELOV3]in the production of sebaceous lipids. The results were compared withcontrol mice treated with vehicle alone: FIG. 4 shows that among thethree structurally related compounds tested (NSA-2, NSA-8 and NSA-9)only NSA-2 induced major inhibition of the mRNA expression encoding thethree enzymes, whereas NSA-8 had a slightly stimulating effect whilstNSA-9 had no significant effect. NSA-2 strongly inhibited in mice theexpression of the genes of key enzymes involved in the production oflipids characteristic of sebum such as AWAT1, ELOVL3, and FADS2, whichat least partly accounts for the sebosuppressive effects of thiscompound. The degree of specific gene suppression seen with NSA-2 isequivalent to that seen with the structurally unrelated compound NSA-3(TCDD).

The Relationship Between 3-Phenyl-1H-Benzo[f]Chromen-1-One Dose andSebosuppressive Effects

The dosing regimen of NSA-2 which achieves significant suppression ofsebogenesis must be carefully defined, in particular to ensure goodtolerability and prevent the theoretical risk onset of cysts of MADISHtype. Recall again, TCDD can act as a pM agonist of AhR and has ahalf-life measurable in years. In contrast, NSA-2 has a half-life inrodents reported at less than 45 mins (Adedoyin et al. (1993)).

In addition, even at 10M, NSA-2, as measured in the standard EROD assay,only activates AhR to 10-20% of the level of induction seen with 0.01 μMTCDD. These essential observations were made in several phases asfollows:

Dose Effect in Mice:

Preliminary studies showed good tolerability of NSA-2 at all dosestested and activity in the sequential in vive activation tests in micedescribed above. For these dose-effects tests, C57BL/6 mice were treatedfor three to five weeks, five days per week on the ears and in threeconcentrations, namely 0.1, 0.5, and 1% of NSA-2. The sebosuppressiveeffect was analyzed at the third week although expression thereof startsafter one week. FIGS. 5-8 show very marked effects which few substancesother than TCDD are able to induce in this model both regarding thetotal number of active sebaceous glands (FIG. 8), the relative surfacesoccupied by the sebaceous glands (FIG. 5) and the ratios betweennon-differentiated, differentiated and mature sebocytes (FIG. 6 and FIG.7).

In particular, a reduction in the differentiated compartment of thegland was observed (FIG. 6 and FIG. 7), in good agreement with thesuppressive effect on the genes encoding sebaceous lipogenesis enzymes(FIG. 4) since the differentiation of the sebocyte is defined bycytoplasmic lipid accumulation. Drug tolerability, in the absence ofMADISH-type cyst production, was assessed after five weeks' treatment.No cystic lesion were observed with NSA-2 at any concentration tested.The blood assay of 3-phenyl-1H-benzo[f]chromen-1-one by HPLC in thesemice did not detect any measurable level (5 nM sensitivity).

Tolerability and Effect of 3-phenyl-1H-benzo[f]chromen-1-one in Man:

-   -   a) A stable 0.5% formulation of        3-phenyl-1H-benzo[f]chromen-1-one was defined.    -   b) Formulation: 3-phenyl-1H-benzo[f]chromen-1-one 0.5 g/100 ml        in ethanol/PEG 400 (1:1).    -   c) Solvents: Ethanol EMSURE® Merck catalog number 1.00983, batch        K42754183.    -   d) Polyethylene Glycol (PEG) 400, Fluka catalog number 81 170,        batch 260154 286 or PEG 400 Aldrich catalog number 202398.    -   e) Stability: No degradation products were observed six months        after preparation.    -   f) Use in man: The formulation was applied once per day to the        face in eleven patients suffering from intense seborrhea and not        eligible for oral treatment with Isotretinoin, six with acne,        four with rosacea and one with seborrheic dermatitis.    -   g) No side effects were noted. In particular no clinical signs        suggesting the onset of microcysts. This provides confirmation        in humans of the safety of topical        3-phenyl-1H-benzo[f]chromen-1-one. This tolerability in man        therefore amounts to original data of primary importance.    -   h) The use of Sebutape® (CUDerm) patch test, to determine the        amount of sebum produced in six individuals after treatment,        indicated a level corresponding to the normal sebum production        range.

CONCLUSION

This invention allows the rapid selection of candidate sebosuppressivemolecules for therapeutic use in treating or preventing acne, seborrheicdermatitis and rosacea.

Persons skilled in the art will easily appreciate that the method of theinvention could be implemented, without departing from the scope of theinvention, in another laboratory mammal other than the C57BL/6 mousestrain, provided that the described activation sequence is reproduced.

The discrepancy between in vitro measurements of receptor activation andin vivo effects may reflect kinetic elements particular to the transportand diffusion of each molecule into the relevant tissues. The methodwhich is the subject of this invention is therefore the first everdescribed which allows investigation of the specific targeting ofdifferent stages of the sebaceous cells within skin tissue by potentialtherapeutic molecules.

All of the ligands able to induce a significant sebosuppressive effectat the clinical level are those which, as early as the first week oftreatment in mice, induced Cyp1A1 staining which exceeded Stage 2. Themost active ligands reached Stage 4 as early as the first week oftreatment (FIG. 3).

During the above-described tests, the 3-phenyl-1H-benzo[f]chromen-1-oneselected after the in vivo tests according to the invention, confirmedits capacity for use as sebosuppressive treatment in the form of atopical application in the human being.

LIST OF REFERENCES

-   1. J. Abel et al. (2010) “An introduction to the molecular basics of    aryl hydrocarbon receptor biology” Biol. Chem., 391, 1235-1248-   2. T. Ikuta et al. (2010) “B lymphocyte-induced maturation protein 1    is a novel target gene of aryl hydrocarbon receptor” J. Derm. Sci.    2010 June, 58 (3), 211-216.-   3. PCT International Application Publication No. WO 2004/041758-   4. PCT International Application Publication No. WO 2007/128725-   5. U.S. Patent Application Publication No. 2009/0028804 A1-   6. P. K. Mandal (2005) “Dioxin: a review of its environmental    effects and its aryl hydrocarbon receptor biology” J Comp Physiol    B., 175(4), 221-230-   7. J. H. Saurat et al. (2012), “The cutaneous lesions of dioxin    exposure: Lessons from the poisoning of V. Yushchenko” Toxicological    Sciences, 125, 310-317-   8. U.S. Patent Application Publication No. 2010/0324109 A1-   9. G. He et al. (2011), “Third-generation Ah receptor-responsive    luciferase reporter plasmids: amplification of dioxin-responsive    elements dramatically increases CALUX bioassay sensitivity and    responsiveness Toxicol. Sci., 123(2), 511-522-   10. G. Zamaratskaia and V. Zlabek (2009) “EROD and MROD as Markers    of Cytochrome P450 1A Activities in Hepatic Microsomes from Entire    and Castrated Male Pigs” Sensors, 9, 2134-2147-   11. Behnisch et al. (2001), “Bioanalytical screening methods for    dioxins and dioxin-like compounds a review of bioassay/biomarker    technology” Environ. Int. 27(5), 413-439-   12. Jensen et al. (2009), “Lrigl Expression Defines a Distinct    Multipotent Stem Cell Population in Mammalian Epidermis” Cell Stem    cell, 4(5), 427-439-   13. C. Niemann and V. Horsley et al. (2012), “Development and    homeostasis of the Sebaceous Gland” Semin. Cell. Dev. Biol., 23(8),    928-936-   14. K. R. Smith and D. M. Thiboutot (2008) “Sebaceous gland lipids:    friend or foe?” The Journal of Lipid Research, 49, 271-281-   15. Miyazaki et al. (2001), “Targeted Disruption of Stearoyl-CoA    Desaturasel Gene in Mice Causes Atrophy of Sebaceous and Meibomian    Glands and Depletion of Wax Esters in the Eyelid” Am. Soc. Nutr.    Sci., 131, 2260-2268-   16. G. Kaya and J. H. Saurat (2007) “Dermatoporosis—A chronic    cutaneous insufficiency/fragility syndrome: Cinico-pathological    features, mechanisms, prevention and potential treatments”    Dermatology, 215(4), 284-294-   17. K. Monostory et al. (2009) “Hormonal regulation of CYP1A    expression” Drug Metab. Rev., 41(4), 547-572-   18. D. W. Nebert and T. P. Dalton (2006), “The role of cytochrome    P450 enzymes in endogenous signalling pathways and environmental    carcinogenesis” Nat. Rev. Cancer, 2006, 6(12), 947-960.-   19. S. F. Zhou et al. (2009), “Polymorphism of human cytochrome P450    enzymes and its clinical impact” Drug Metab. Rev. 2009, 4(2),    82-295.-   20. P. Milde et al (1991) “Expression of 1,25-dihydroxyvitamin D3    receptors in normal and psoriatic skin” J. Invest Dermatol. 97(2),    230-9.-   21. J. Reichrath et al., (1997) “Expression of retinoid-X receptors    and retinoic acid receptors in normal human skin: an    immunchistological evaluation” The Histochemical Journal 29(2),    127-133-   22. M. J. Rowe et al. (2008) “Illuminating role of CYP1A1 in skin    function” J. Invest. Derm., 128, 1866-1868-   23. D. Frances and C. Niemann (2012), “Stem cell dynamics in    sebaceous gland morphogenesis in mouse skin” Dev. Biol., 363 (1),    138-146-   24. H. Dobrev (2007) “Clinical and instrumental study of the    efficacy of a new sebum control cream” Journal of Cosmetic    Dermatology, 6(2), 113-118-   25. A. Adedoyin et al., (1993) “Time-dependent disposition of    beta-naphthoflavone in the rat.” Pharmaceut Res, 10, 35-43

What is claimed:
 1. A method of treating acne in a subject whichcomprises topically and periodically applying to the subject's acne acomposition comprising 3-phenyl-1-benzo[f]chromen-1-one and apharmaceutically acceptable carrier, wherein the3-phenyl-1-benzo[f]chromen-1-one is present in an amount effective totreat the subject's acne.
 2. The method of claim 1, wherein the3-phenyl-1-benzo[f]chromen-1-one is present in the composition at aconcentration between about 0.005% and about 5% by weight.
 3. The methodof claim 2, wherein the 3-phenyl-1-benzo[f]chromen-1-one is present inthe composition at a concentration between about 0.1% and about 2.5% byweight.
 4. The method of claim 3, wherein the3-phenyl-1-benzo[f]chromen-1-one is present in the composition at aconcentration of about 0.1% by weight.
 5. The method of claim 3, whereinthe 3-phenyl-1-benzo[f]chromen-1-one is present in the composition at aconcentration of about 0.25% by weight.
 6. The method of claim 3,wherein the 3-phenyl-1-benzo[f]chromen-1-one is present in thecomposition at a concentration of about 0.5% by weight.
 7. The method ofclaim 3, wherein the 3-phenyl-1-benzo[f]chromen-1-one is present in thecomposition at a concentration of about 1% by weight.
 8. The method ofclaim 3, wherein the 3-phenyl-1-benzo[f]chromen-1-one is present in thecomposition at a concentration of about 2% by weight.
 9. The method ofclaim 2, wherein the 3-phenyl-1-benzo[f]chromen-1-one is present in thecomposition at a concentration of about 5% by weight.
 10. The method ofclaim 1, wherein the pharmaceutically acceptable carrier comprisesethanol.
 11. The method of claim 1, wherein the pharmaceuticallyacceptable carrier comprises polyethylene glycol having an averagemolecular weight between 200 g/mol and 1000 g/mol.
 12. The method ofclaim 11, wherein the polyethylene glycol has an average molecularweight of about 400 g/mol.
 13. The method of claim 1, 11 or 12, whereinthe pharmaceutically acceptable carrier comprises a mixture of ethanoland polyethylene glycol in a ratio from 5:1 to 1:5 by volume.
 14. Themethod of claim 13, wherein the ratio is between 2:1 and 1:2 by volume.15. The method of claim 14, wherein the ratio is about 1:1 by volume.16. The method of claim 1, wherein the pharmaceutical composition is asolution and comprises 3-phenyl-1-benzo[f]chromen-1-one at aconcentration between 0.005 g and 1.0 g 3-phenyl-1-benzo[f]chromen-1-oneper 100 mL of the composition and the pharmaceutically acceptablecarrier comprises a mixture of ethanol and polyethylene glycol having anaverage molecular weight of about 400 g/mol in a ratio of about 1:1 byvolume.
 17. The method of claim 16, wherein the concentration of3-phenyl-1-benzo[f]chromen-1-one is between 0.05 g and 0.5 g.
 18. Themethod of claim 16, wherein the 3-phenyl-1-benzo[f]chromen-1-one is at aconcentration of about 0.5 g, the polyethylene glycol has an averagemolecular weight of about 400 g/mol and the mixture of ethanol andpolyethylene glycol is in a ratio of about 1:1 by volume.
 19. The methodof claim 1, wherein the pharmaceutically acceptable carrier furthercomprises one or more of an alcohol, an anti-bacterial agent, apreservative, and a chelating agent.
 20. The method of any one of claims1-19, wherein the pharmaceutical composition is a lotion, gel, cream,ointment, foam, solution, suspension, dispersion or impregnateddressing.
 21. The method of claim 1, wherein the acne is facial acne.22. The method of claim 1, wherein the acne is associated withPropionibacterium acnes.
 23. The method of claim 1, wherein the acne isassociated with a high sebum secretion rate.
 24. The method of claim 1,wherein 3-phenyl-1-benzo[f]chromen-1-one is topically applied daily. 25.The method of claim 1 or 24, wherein 3-phenyl-1-benzo[f]chromen-1-one istopically applied only at night.
 26. The method of claim 1, wherein3-phenyl-1-benzo[f]chromen-1-one is topically applied twice or threetimes daily.
 27. The method of claim 1, wherein3-phenyl-1-benzo[f]chromen-1-one is topically applied every other day.28. The method of claim 1, wherein 3-phenyl-1-benzo[f]chromen-1-one istopically applied weekly.
 29. A method of treating a skin conditionassociated with abnormal sebum secretion or abnormal sebaceous glandfunction in a subject which comprises topically and periodicallyapplying to an area of subject's skin affected by the skin condition acomposition comprising 3-phenyl-1-benzo[f]chromen-1-one and apharmaceutically acceptable carrier, wherein the3-phenyl-1-benzo[f]chromen-1-one is present in an amount effective totreat the skin condition.
 30. The method of claim 29, wherein the skincondition is oily skin, oily hair, shiny or greasy-looking skin,hyperseborrhea, seborrheic dermatitis, rosacea, sebaceous hyperplasia orsebaceous carcinoma.
 31. The method of claim 30, wherein the skincondition is seborrheic dermatitis.
 32. The method of claim 30, whereinthe skin condition is rosacea.
 33. The method of claim 30, wherein theskin condition is hyperseborrhea.
 34. The method of claim 30, whereinthe skin condition is sebaceous hyperplasia.
 35. The method of claim 30,wherein the skin condition is sebaceous carcinoma.
 36. The method ofclaim 29, wherein the 3-phenyl-1-benzo[f]chromen-1-one is present in thecomposition at a concentration between about 0.005% and about 5% byweight.
 37. The method of claim 36, wherein the3-phenyl-1-benzo[f]chromen-1-one is present in the composition at aconcentration between about 0.1% and about 2.5% by weight.
 38. Themethod of claim 37, wherein the 3-phenyl-1-benzo[f]chromen-1-one ispresent in the composition at a concentration of about 0.1% by weight.39. The method of claim 37, wherein the 3-phenyl-1-benzo[f]chromen-1-oneis present in the composition at a concentration of about 0.25% byweight.
 40. The method of claim 37, wherein the3-phenyl-1-benzo[f]chromen-1-one is present in the composition at aconcentration of about 0.5% by weight.
 41. The method of claim 37,wherein the 3-phenyl-1-benzo[f]chromen-1-one is present in thecomposition at a concentration of about 1% by weight.
 42. The method ofclaim 37, wherein the 3-phenyl-1-benzo[f]chromen-1-one is present in thecomposition at a concentration of about 2% by weight.
 43. The method ofclaim 36, wherein the 3-phenyl-1-benzo[f]chromen-1-one is present in thecomposition at a concentration of about 5% by weight.
 44. The method ofclaim 29, wherein the pharmaceutically acceptable carrier comprisesethanol.
 45. The method of claim 29, wherein the pharmaceuticallyacceptable carrier comprises polyethylene glycol having an averagemolecular weight between 200 g/mol and 1000 g/mol.
 46. The method ofclaim 29, wherein the polyethylene glycol has an average molecularweight of about 400 g/mol.
 47. The method of claim 29, 45 or 46, whereinthe pharmaceutically acceptable carrier comprises a mixture of ethanoland polyethylene glycol having an average molecular weight of about 400g/mol in a ratio from 5:1 to 1:5 by volume.
 48. The method of claim 47,wherein the ratio is between 2:1 and 1:2 by volume.
 49. The method ofclaim 48, wherein the ratio is about 1:1 by volume.
 50. The method ofclaim 29, wherein the pharmaceutical composition is a solution andcomprises 3-phenyl-1-benzo[f]chromen-1-one at a concentration between0.005 g and 1.0 g 3-phenyl-1-benzo[f]chromen-1-one per 100 mL of thecomposition and the pharmaceutically acceptable carrier comprises amixture of ethanol and polyethylene glycol having an average molecularweight of about 400 g/mol in a ratio of about 1:1 by volume.
 51. Themethod of claim 50, wherein the concentration of3-phenyl-1-benzo[f]chromen-1-one is between 0.05 g and 0.5 g.
 52. Themethod of claim 50, wherein the 3-phenyl-1-benzo[f]chromen-1-one is at aconcentration of about 0.5 g, the polyethylene glycol has an averagemolecular weight of about 400 g/mol and the mixture of ethanol andpolyethylene glycol is in a ratio of about 1:1 by volume.
 53. The methodof claim 29, wherein the pharmaceutically acceptable carrier furthercomprises one or more of an alcohol, an anti-bacterial agent, apreservative, and a chelating agent.
 54. The method of any one of claims29-53, wherein the pharmaceutical composition is a lotion, gel, cream,ointment, foam, solution, suspension, dispersion or impregnateddressing.
 55. The method of claim 29, wherein the area of the subject'sskin affected by the skin condition is on the face.
 56. The method ofclaim 29, wherein the skin condition is associated withPropicnibacterium acnes.
 57. The method of claim 29, wherein the skincondition is associated with a high sebum secretion rate.
 58. The methodof claim 29, wherein 3-phenyl-1-benzo[f]chromen-1-one is topicallyapplied daily.
 59. The method of claim 29 or 58, wherein3-phenyl-1-benzo[f]chromen-1-one is topically applied only at night. 60.The method of claim 29, wherein 3-phenyl-1-benzo[f]chromen-1-one istopically applied twice or three times daily.
 61. The method of claim29, wherein 3-phenyl-1-benzo[f]chromen-1-one is topically applied everyother day.
 62. The method of claim 29, wherein3-phenyl-1-benzo[f]chromen-1-one is topically applied weekly.
 63. Acomposition comprising 3-phenyl-1-benzo[f]chromen-1-one and apharmaceutically acceptable carrier, wherein the3-phenyl-1-benzo[f]chromen-1-one is present at a concentration betweenabout 0.005% and about 5% by weight.
 64. The composition of claim 63,wherein the 3-phenyl-1-benzo[f]chromen-1-one is present at aconcentration between about 0.1% and about 2.5% by weight.
 65. Thecomposition of claim 64, wherein the 3-phenyl-1-benzo[f]chromen-1-one ispresent at a concentration of about 0.1% by weight.
 66. The compositionof claim 64, wherein the 3-phenyl-1-benzo[f]chromen-1-one is present ata concentration of about 0.25% by weight.
 67. The composition of claim64, wherein the 3-phenyl-1-benzo[f]chromen-1-one is present at aconcentration of about 0.5% by weight.
 68. The composition of claim 64,wherein the 3-phenyl-1-benzo[f]chromen-1-one is present at aconcentration of about 1% by weight.
 69. The composition of claim 64,wherein the 3-phenyl-1-benzo[f]chromen-1-one is present at aconcentration of about 2% by weight.
 70. The composition of claim 63,wherein the 3-phenyl-1-benzo[f]chromen-1-one is present at aconcentration of about 5% by weight.
 71. The composition of claim 63,wherein the pharmaceutically acceptable carrier comprises ethanol. 72.The composition of claim 63, wherein the pharmaceutically acceptablecarrier comprises polyethylene glycol having an average molecular weightbetween 200 g/mol and 1000 g/mol.
 73. The composition of claim 72,wherein the polyethylene glycol has an average molecular weight of about400 g/mol.
 74. The composition of claim 63, 72 or 73, wherein thepharmaceutically acceptable carrier comprises a mixture of ethanol andpolyethylene glycol in a ratio from 5:1 and 1:5 by volume.
 75. Thecomposition of claim 74, wherein the ratio is between 2:1 and 1:2 byvolume.
 76. The composition of claim 75, wherein the ratio is about 1:1by volume.
 77. The composition of claim 63, wherein the pharmaceuticalcomposition is a solution and comprises 3-phenyl-1-benzo[f]chromen-1-oneat a concentration between 0.005 g and 1.0 g3-phenyl-1-benzo[f]chromen-1-one per 100 mL of the composition and thepharmaceutically acceptable carrier comprises a mixture of ethanol andpolyethylene glycol having an average molecular weight of about 400g/mol in a ratio of about 1:1 by volume.
 78. The composition of claim77, wherein the concentration of 3-phenyl-1-benzo[f]chromen-1-one isbetween 0.05 g and 0.5 g.
 79. The composition of claim 77, wherein the3-phenyl-1-benzo[f]chromen-1-one is at a concentration of about 0.5 g,the polyethylene glycol has an average molecular weight of about 400g/mol and the mixture of ethanol and polyethylene glycol is in a ratioof about 1:1 by volume.
 80. The composition of claim 63, wherein thepharmaceutically acceptable carrier further comprises one or more of analcohol, an anti-bacterial agent, a preservative, and a chelating agent.81. The composition of any one of claims 63-80, wherein thepharmaceutical composition is a lotion, gel, cream, ointment, foam,solution, suspension, dispersion or impregnated dressing.